The advances in sequencing technology have provided a superior alternative for the detection of cancer mutations. Next-generation sequencing (NGS) is a cutting age technology with a superior resolution capable of reaching down to the single nucleotide level. However, the application of NGS in the analysis of cancer mutations from circulating plasma DNA (ctDNA), which may carry a small amount of tumor circulating DNA (ctDNA A), is still at an early stage. This is due to the fact that the DNA level in plasma is extremely low and that there are not many reliable methods to amplify a small amount of DNA from the plasma. Conventional DNA amplification by polymerase chain reaction (PCR) utilizes one or more primer “pairs” to define the boundaries of a target region(s) and prime the synthesis by thermophilic DNA polymerase.
The strategy of using paired primers has an intrinsic disadvantage when being applied to full-length amplification of linear DNA because it is essential for the termini of each target DNA fragment to ligate to two different adapters, which will enable the binding of different PCR primers for cyclic DNA amplification. Since every terminus has equal chance to bind to either adapter, half of the DNA fragments are ligated to only one type of adapter, resulting in a 50% potential loss of sequence information because of the inability of such fragments to be amplified at exponential rate and thus will become mistakenly underrepresented in sequencing and downstream analysis. This problem can be troublesome when it comes to NGS analysis of low abundance DNA molecules such as those in single-cell transcriptome or those in body fluid specimens such as plasma, where the majority of target DNA or RNA species are far below the minimal level required for sequencing library construction or for quantification by conventional laboratory instruments. This problem can become even more severe when disease diagnosis is concerned, because a huge number of clinical DNA samples are examined on daily basis and significant portion of these samples are of low quantity or low quality due to various reasons such as paraffin-embedding, long or improper storage, or at a small volume or a low concentration. Therefore, an efficient and nonselective DNA amplification method which is able to target and amplify “all” DNA/RNA molecules, without sequence discrimination, in the sample is strongly desired.